The long-term objective of this research is to determine the origin of extracellular adenosine in adipose tissue. This locally-produced chemical promotes energy storage and reduces lipid mobilization. These metabolic effects suggest adenosine as a potential contributor to the etiology of obesity. The source of extracellular adenosine in adipose tissue is unknown. The proposed research project will evaluate intracellular cyclic AMP as a source of extracellular adenosine. Specific research aims are 1) to characterize cyclic AMP efflux from adipocytes, 2) to demonstrate extracellular cyclic AMP metabolism to AMP, and 3) to demonstrate extracellular AMP metabolism to adenosine. To meet these aims, we will use isolated adipocytes and isolated adipocyte membrane fractions from miniature swine. Advantages of the miniature swine model are its similarities to the human, its abundance of adipose tissue, and our ability to biopsy tissue without killing the animal. Experiments for aim 1) will use cells isolated from subcutaneous adipose tissue of 3 mo-old swine to measure the time course and energy- and temperature-dependence of cyclic AMP efflux from intact adipocytes. The unidirectionality and inhibitor sensitivity of this process will also be determined. Experiments for aim 2) will characterize the phosphodiesterase enzyme responsible for the conversion of cyclic AMP to AMP in isolated adipocyte plasma membranes, and measure ecto-phosphodiesterase activity of intact adipocytes. Experiments for aim 3) will characterize the 5'-nucleotidase enzyme responsible for the conversion of AMP to adenosine in isolated adipocyte plasma membranes, and measure ecto-5'-nucleotidase activity of intact adipocytes. Examining cyclic AMP as a precursor of extracellular adenosine in adipose tissue will extend our knowledge of adipose tissue as an endocrine organ, deepen our understanding of how adipose tissue regulates its own metabolism, and broaden the scope of obesity research.